None of the references described herein are admitted to be prior art to the claimed invention.
A target nucleic acid sequence can be detected by various methods using detection probes designed to preferentially hybridize to the target sequence over other sequences that may be present in a sample. Examples of target sequences include sequences initially present in a sample or produced as part of an amplification procedure.
Examples of detection probes include oligonucleotides and derivatives thereof able to preferentially hybridize to a target nucleic acid containing a target nucleic acid sequence over other nucleic acids that may be present in a sample. Hybridization of detection probes to target nucleic acid sequences results in the formation of detectable probe:target hybridization complexes under appropriate conditions.
Detecting detectable probe:target hybridization complexes is facilitated using a labeled detection probe. Different labels and assay formats can be used to detect the presence or amount of an analyte in a sample. Examples of detectable labels include radioisotopes, fluorescent molecules, chemiluminescent molecules, chromophors, enzymes, enzyme substrates and ligands. Examples of references describing the detection of nucleic acid using fluorescent and chemiluminescent molecules include Arnold et al., U.S. Pat. Nos. 5,283,174 and Becker et al. 5,731,148, both of which are hereby incorporated by reference herein.
To facilitate detection of a target nucleic acid sequence, the number of target sequences in a sample can be increased using nucleic acid amplification techniques. Nucleic acid amplification involves the enzymatic synthesis of nucleic acid containing a sequence complementary to a nucleic acid sequence being amplified. Nucleic acid amplification can be performed using different techniques such as those involving transcription-based amplification, the polymerase chain reaction (PCR), ligase chain reaction (LCR) and strand displacement amplification (SDA).
Transcription-based amplification of a nucleic acid sequence generally employs an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a promoter-template complementary oligonucleotide. The promoter-template complementary oligonucleotide contains a 5' sequence recognized by an RNA polymerase and a 3' sequence that hybridizes to a template nucleic acid in a location 3' of a sequence sought to be amplified. After hybridization of the promoter-template complementary oligonucleotide to the template, a double-stranded promoter is formed upstream from the target nucleic acid sequence. Double-stranded promoter formation generally involves DNA polymerase activity. Generally, a second oligonucleotide primer is employed to facilitate double-stranded promoter formation.
Transcription-based amplification involves the binding of an RNA polymerase to a promoter region that is usually double-stranded. The RNA polymerase proceeds downstream from the promoter region and synthesizes ribonucleic acid in a 5' to 3' direction. Multiple RNA transcripts are produced by transcription-based amplification using a single template.
Different formats can be employed for performing transcription-based amplification. Examples of different formats are provided in publications such as Burg et al., U.S. Pat. Nos. 5,437,990; Kacian et al., 5,399,491; Kacian et al., 5,554,516; McDonough et al., 5,766,849; Ryder et al., 5,786,183; Malek et al., 5,130,238; Kacian et al., International Application No. PCT/US93/04015, International Publication No. WO 93/22461; Gingeras et al., International Application No. PCT/US87/01966, International Publication No. WO 88/01302; Gingeras et al., International Application No. PCT/US88/02108, International Publication No. WO 88/10315; Davey and Malek, European Application No. 88113948.9, European Publication No. 0 329 822 A2; and Urdea, International Application No. PCT/US91/00213, International Publication No. WO 91/10746. (Each of these references is hereby incorporated by reference herein.)
PCR amplification is described by Mullis et al., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159, and in Methods in Enzymology, 155:335-350 (1987). (Each of these references is hereby incorporated by reference herein.)
An example of LCR is described in European Patent Publication No. 320 308, which is hereby incorporated by reference herein. LCR uses at least four separate oligonucleotides. Two of the oligonucleotides hybridize to a nucleic acid template so that the 3' end of one oligonucleotide and the 5' end of the other oligonucleotide are positioned for ligation. The hybridized oligonucleotides are then ligated forming a full-length complement to the target nucleic acid sequence. The double-stranded nucleic acid is then denatured, and third and fourth oligonucleotides are hybridized to the complementary strand and joined together. Amplification is achieved by further cycles of hybridization, ligation, and denaturation, producing multiple copies of the target nucleic acid sequence and the sequence complementary to the target nucleic acid sequence.
SDA is an isothermal amplification reaction based on the ability of a restriction enzyme to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and on the ability of a DNA polymerase to initiate replication at the nick and displace a downstream non-template strand. (See, e.g., Walker, PCR Methods and Applications, 3:25-30 (1993), Walker et al., Nucleic Acids Res., 20:1691-1996 (1992), and Walker et al., Proc. Natl. Acad. Sci. 89:392-396 (1991). (Each of these references is hereby incorporated by reference herein.) The steps used in generating fragments for carrying out autocatalytic SDA amplification are indicated to be adaptable for generating fragments for transcription-based amplification or amplification carried out using Q-beta technology (Walker et al., Nucleic Acids Res., 20:1691-1696 (1992), which is hereby incorporated by reference herein.